Analytical device with lightguide illumination of capillary and microgroove arrays

ABSTRACT

An analytical cell including a lightguide with a plurality of conduits filled with a migration medium. The medium, the lightguide and a surrounding medium have refractive indices selected such that light entering the lightguide is internally reflected within the lightguide to provide substantially uniform illumination of the conduits.

This application is a continuation of and claims priority to Ser. No.10/028,257, filed Dec. 19, 2001 now U.S. Pat. No. 7,189,361, the entirecontent of which is incorporated herein by reference.

TECHNICAL FIELD

This invention relates to methods and apparatus for detecting biologicalmacromolecules.

BACKGROUND

Techniques for analyzing biological macromolecules, such as, forexample, nucleic acids and proteins, have become increasingly importantin the fields of medicine and genetics. One well accepted technique foranalyzing biomolecules is gel electrophoresis. In gel electrophoresis avoltage is applied across at least one linear dimension of a medium,typically a liquid buffer or a polymer gel. A sample tagged with afluorophore is introduced to the medium, and components of the sampleseparate under the influence of the applied electric field according totheir respective electric mobilities. The fluorescently labeledcomponents migrate down the linear dimension of the medium past astation where they are illuminated by a laser beam. Stimulatedfluorescent emission from the illuminated components is captured by adetector as a function of time, producing an electropherogram thatencodes the analytical information of interest.

Electrophoresis devices are available in a variety of formats.Traditionally, separations are performed in a medium made ofcross-linked polymer matrix formed as a gel sheet, or slab gel, betweentwo glass plates. To enable higher applied voltages, remove heatgenerated by electrophoretic currents, and provide higher throughput,the medium may be confined to narrow glass capillary tubes. Microgroovesfabricated into a planar, laminated substrate of glass or plastic havealso been used as conduits for the medium.

In a high throughput analytical device, the capillaries or microgrooves,referred to herein as sample conduits, are arranged in substantiallyplanar arrays so that many samples may be processed at the same time.The array format is most efficient when a single laser, or a smallnumber of lasers, is used to illuminate the capillaries or microgroovesin the array. Since the medium in each conduit absorbs only a tinyfraction of the laser power, most devices utilize an arrangement inwhich the optical axis of the laser beam output is substantiallycoplanar with and normal to the longitudinal axes of the conduits. Asingle laser beam, or, in some cases opposed dual beams, impinge normalto the wall of the first conduit in a substantially planar array,illuminate the fluorescently labeled sample therein, exit the firstconduit, propagate to the second conduit, and so forth. This techniquehas been generally successful for arrays with a small number ofconduits, but becomes increasingly unworkable as the number of conduitsin the array is increased. The variety of materials in the beam path(for example, glass, medium, air), each having its own index ofrefraction, as well as the multiplicity of surfaces, creates anextremely complex optical system. Reflection and refraction of the beamat the multiple surfaces diverts the beam from a direct passage thoughthe conduits, which makes efficient and uniform delivery of the light toeach conduit problematic.

The need for relative uniformity of illumination stems from theeconomical practice of using a single detector (or an array of identicaldetector elements) for measuring signal from each conduit of the planararray. As such, the signal from each conduit, proportional to theintensity of excitation, is detected with the same level of sensitivityand dynamic range. In this arrangement, nonuniform illumination woulddictate undesirable trade-offs. For example, adjusting the intensity ofthe laser beam to achieve maximal sensitivity in a relatively poorlyilluminated conduit could lead to detector saturation by signals ofother, better illuminated conduits, thereby limiting the dynamic rangeof the better illuminated conduits. Therefore, array performance isoptimized by ensuring that all conduits receive the same intensity ofexcitation light.

In each of these systems, the array of conduits is treated as asequential optical system in which all or most of the light energypassing out of one conduit impinges on the next successive conduit inthe array. These systems are extremely sensitive to optical misalignmentand must be assembled to extremely high tolerances, so manufacturingyields would be expected to be quite low. In addition, this delicateoptical system would be easily misaligned if repeatedly handled andinstalled in an analytical device.

The treatment of conduits as optical elements also places constraints intheir geometry, depending on the optical properties of the materialsused. For example, for capillaries in a close packed configuration, theratio of the inner and outer diameters of the capillaries are restrictedto a specific range, depending on the refractive indices of thecapillary walls, the enclosed medium, and the surrounding medium.Capillaries with dimensions outside these ranges will fail toeffectively transmit the beam from one capillary to the next. Opticalalignment is not as significant a problem for microgrooves arrays, whichmay be precisely laid out equidistant from one another on a substrate.However, embossing and chemical etching procedures used to form themicrogrooves in the substrate create beveled walls that are notperpendicular to the plane of the array or to the light source. Whensealed with a coversheet and filled with a polymer medium, eachmicrogrooves can form a prism-like optical structure that cumulativelycauses the beam to deflect out of plane, leaving a majority of themicrogrooves insufficiently illuminated.

Previous proposals for array illumination have made unacceptablecompromises in illumination intensity or uniformity, or have demandedprohibitive requirements in optical alignment.

SUMMARY

In one embodiment, the invention is an analytical cell for detection ofan analyte. The cell includes an elongate lightguide having an array ofconduits extending therethough. The conduits are configured to support amigration medium. The lightguide and its surrounding medium haverefractive indices selected such that light entering the lightguide isinternally reflected within the lightguide to illuminate the conduits.

In a second embodiment, the invention is an analytical cell including acover on a substrate. The substrate includes an array of substantiallyparallel grooves, wherein the grooves are substantially coplanar and areconfigured to support a migration medium. The migration medium, thesubstrate, the cover and the surrounding medium have refractive indicesselected such that a lightguide is formed when the cover is placed onthe substrate, and light entering the lightguide is totally internallyreflected within the lightguide to illuminate the grooves.

In a third embodiment, the invention is an analytical device includingan elongate lightguide. The lightguide includes a substrate with anarray of substantially parallel grooves configured to support amigration medium, wherein the grooves are substantially coplanar andhave a longitudinal axis in a first direction, and a cover on thesubstrate. A light source is placed outside the lightguide, wherein thesource emits a light beam with an optical axis substantially coplanarwith and normal to the longitudinal axes of the grooves. The migrationmedium, the substrate, the cover and a medium surrounding the substratehave refractive indices selected such that light emitted by the lightsource is totally internally reflected within the lightguide toilluminate the grooves.

In a fourth embodiment, the invention is an assay method including:

(a) providing an analytical cell including: (1) a substrate with aplurality of substantially parallel grooves, wherein the grooves aresubstantially coplanar, are configured to support a migration medium,and have longitudinal axes in a first direction, and (2) a cover on thesubstrate; wherein the migration medium, the substrate, the cover and amedium surrounding the substrate have refractive indices selected suchthat a lightguide is formed when the cover is placed on the substrate,and light entering the lightguide is internally reflected within thelightguide to illuminate the grooves;

(b) placing a sample on the migration medium in a groove, wherein thesample comprises a fluorescently labeled analyte;

(c) applying an electric field across the first direction to move theanalyte in the groove;

(d) illuminating the lightguide with a light beam having an optical axisalong a second direction substantially coplanar with the plane of thegrooves and normal to the first direction, wherein the light enteringthe lightguide is totally internally reflected within the lightguide toilluminate at least a portion of each groove; and

(e) detecting an emission from the analyte.

In a fifth embodiment, the invention is an analytical cell including asolid lightguide. The lightguide includes a first wall with a firstinterior surface, a second wall with a second interior surface, whereinthe second wall is opposite the first wall, and the second interiorsurface faces the first interior surface, a third wall with a thirdinterior surface, and a fourth wall opposite the third wall, and asurrounding medium adjacent at least one of the walls. The lightguidefurther includes a plurality of capillaries configured to support amigration medium, wherein the capillaries are fixed in an array at leastpartially enclosed within the lightguide, wherein the longitudinal axesof the capillaries are substantially parallel and coplanar. Themigration medium, the capillaries, the lightguide and the surroundingmedium have refractive indices selected such that light entering thelightguide is internally reflected within the lightguide at the interiorsurfaces to illuminate the capillaries.

In a sixth embodiment, the invention is an analytical cell including alightguide. The lightguide includes a substrate with a plurality ofsubstantially parallel grooves, wherein the grooves are substantiallycoplanar and have a substantially arcuate cross section, and a coverincluding an array of substantially parallel grooves corresponding tothe grooves in the substrate, wherein the grooves in the cover aresubstantially coplanar and have a substantially arcuate cross section. Aplurality of capillaries reside in the grooves between the substrate andthe cover, wherein the capillaries have a substantially circular crosssection, and the longitudinal axes of the capillaries extend in a firstdirection to form a substantially coplanar array, and wherein thecapillaries are configured to support a migration medium. The migrationmedium, the capillaries, the substrate, the cover and a medium borderingthe substrate have refractive indices selected light entering thelightguide from a second direction substantially coplanar with andnormal to the first direction is totally internally reflected within thelightguide to illuminate the array.

In a seventh embodiment, the invention is an analytical device,including a lightguide. The lightguide includes a substrate with aplurality of substantially parallel grooves, wherein the grooves aresubstantially coplanar and have a substantially arcuate cross section,(2) a cover including a plurality of substantially parallel groovescorresponding to the grooves in the substrate, wherein the grooves inthe cover are substantially coplanar and have a substantially arcuatecross section. A plurality of capillaries reside in the grooves betweenthe substrate and the cover, wherein the capillaries have asubstantially circular cross section, and the longitudinal axes of thecapillaries extend in a first direction to form a substantially coplanararray, and wherein the capillaries are configured to support a migrationmedium. A light source is placed outside the lightguide, wherein thelight source emits a beam having an optical axis substantially coplanarwith and normal to the longitudinal axes of the capillaries in thearray. The migration medium, the capillaries, the substrate, the coverand a medium bordering the substrate have refractive indices selectedsuch that light emitted by the light source is totally internallyreflected within the lightguide to illuminate the array.

In an eighth embodiment, the invention is an assay method including:

(1) providing an analytical cell including:

(a) a lightguide including (1) a substrate with a plurality ofsubstantially parallel grooves, wherein the grooves are substantiallycoplanar and have a substantially arcuate cross section, and (2) a covercomprising a plurality of substantially parallel grooves correspondingto the grooves in the substrate, wherein the grooves in the cover aresubstantially coplanar and have a substantially arcuate cross section;

(b) a plurality of capillaries in the grooves between the substrate andthe cover, wherein the capillaries have a substantially circular crosssection, and the longitudinal axes of the capillaries extend in a firstdirection to form a substantially coplanar array, and wherein thecapillaries are configured to support a migration medium;

(2) placing a sample on the migration medium in each capillary in thearray, wherein the sample comprises a fluorescently labeled analyte;

(3) applying an electric field across the first direction to move theanalyte in a capillary in the array;

(4) illuminating the lightguide with a light beam having an optical axisalong a second direction substantially coplanar with the plane of thearray and normal to the first direction, wherein the light entering thelightguide is totally internally reflected within the lightguide toilluminate at least a portion of the array; and

(5) detecting with a detector an emission from the analyte.

In a ninth embodiment, the invention is an analyte separation device forthe detection of one or more fluorescently labeled analytes, including(a) an elongate lightguide; (b) an array of conduits in the lightguide,wherein the conduits are configured to support a migration medium; (c) alight source optically coupled to the lightguide, wherein the lightguidehas a refractive index greater than its surrounding medium such thatlight emitted by the source is totally internally reflected within thelightguide to illuminate the conduits; and (d) a detector opticallycoupled to the conduits.

With the invention, uniform illumination is achieved at a reasonableloss in intensity relative to direct illumination of a single capillary.In addition, the invention is very tolerant of errors in fabrication andoperation, including, for example, misalignment of the light source,misalignment of the conduits in the array, and variations in channelbevel.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic representation in perspective of an analyticaldevice using the analytical cell of the invention.

FIG. 1B is a schematic overhead view of an analytical device using theanalytical cell of the invention.

FIG. 2A is a cross sectional view of an analytical cell of the inventionwith microgrooves.

FIG. 2B is a cross sectional view of a trapezoidal analytical cell ofthe invention with microgrooves.

FIG. 3 is a cross sectional view of an analytical cell of the inventionwith microgrooves, showing the optical path of selected incoming lightrays.

FIG. 4 is a cross sectional view of a two part analytical cell of theinvention with microgrooves.

FIG. 5 is a cutaway, perspective view of an analytical cell of theinvention with capillaries.

FIG. 6 is a cross sectional view of an analytical cell of the inventionwith capillaries.

FIG. 7 is a cross sectional view of an analytical cell of the inventionwith capillaries, showing the optical path of selected incoming lightrays.

FIG. 8 is a cross sectional view of a two part analytical cell of theinvention with capillaries.

FIG. 9 is a cross sectional view of an analytical cell of the inventionwith close-packed capillaries.

FIG. 10 is a cross sectional view of an analytical cell of the inventionwith close-packed, staggered capillaries.

FIG. 11 is a cross sectional view of an analytical cell of the inventionwith capillaries having a non-circular cross sectional shape.

FIG. 12 is a perspective view of a analytical cell of the inventionhaving microgrooves with a square cross sectional shape and adapted toreceive capillaries having a circular cross sectional shape.

FIG. 13A and FIG. 13B are schematic representations of the incominglight beam in an analytical device of the invention.

FIG. 14 is a cross sectional view of an analytical cell of the inventionwith a lens-like face.

FIG. 15 is a cross sectional view of an analytical cell of the inventionwith a grating-like face.

FIG. 16 is a cross sectional view of an analytical cell of the inventionusing two sources of illumination.

FIG. 17 is a plot of relative illumination versus microgrooves numberfor the array of Example 1.

FIG. 18 is a plot of relative illumination versus capillary number forthe array of Example 2.

FIG. 19 is a plot of relative illumination versus capillary number forthe array of Example 2 with non-optimal optical alignment of the lightsource and capillaries.

FIG. 20 is a plot of relative illumination versus capillary number forthe array of Example 2 with variation in the angular spread of theincoming beam.

FIG. 21 is a plot comparing relative illumination versus capillarynumber for the array of Example 2 with that of a similar array havingcapillaries with a square cross sectional shape.

FIG. 22 is a plot of relative illumination versus capillary number forthe array of Example 2 with a reflective third interior surface,compared to an otherwise identical array with a non-reflective interiorsurface, as well as an identical array using dual source illumination.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

FIG. 1A illustrates the major features of an embodiment of an analyticaldevice of the invention. Generally, an analytical device 10 of theinvention includes three principal components: a light source 12, ananalytical cell 14 and a detector 16.

Referring to FIG. 1A, in one embodiment the cell 14 includes a pluralityof conduits 18 with substantially parallel longitudinal axes. Theconduits 18 are arranged in a substantially coplanar array, and arefilled with a migration medium (not shown in FIG. 1A). When afluorescently labeled sample is placed on the migration medium and anelectric field is applied across a direction parallel to thelongitudinal axes of the conduits, components of the sample migratealong the conduits and separate into a series of fluorescently labeledanalytes. When a selected analyte enters the fluorescence detection cell14, a light beam emitted from the light source 12 illuminates the cell14. The beam from the light source 12 has an optical axis generally inthe plane of the conduits 18 and normal to their longitudinal axes. Whenthe light from the source 12 enters the cell 14, the light is totallyinternally reflected within the cell 14 to illuminate each of theconduits 18. The cell 14 acts as a lightguide that retains a substantialportion of the entering light and efficiently delivers it to each of theconduits in the array. Fluorescent emissions from the analyte aredetected by the detector 16 to provide analytical information regardingthe composition of the sample. The detector 16 may include one or moreof the following elements: lenses and optical elements for collectinglight from the cell 14, an aperture for exerting precise control overthe spatial origin of light, diffraction gratings or prisms for spectraldecomposition of the emitted light, and a two-dimensional photodetectorsuch as a charge-coupled device (CCD) camera.

As shown in FIG. 1B, the refractive index of the cell 14 may be selectedwith respect to the surrounding medium to confine the incoming lightrays 13 from the source 12 to a specific volume. The optical intensity(power/unit volume) in this volume is sufficient to illuminate aselected portion of the each conduit 18 in the array and cause theanalytes in that selected portion of each conduit to fluoresce. Thefluorescent emissions 17 from the analytes then exit the illuminatedvolume and are detected by the detector 16 (not shown in FIG. 1B). Theshape and dimensions of the illuminated volume may be controlled tocontain the incoming light to provide an analytical device with adesired array size, throughput and resolution.

Referring to FIG. 2A, a cross-sectional view of an embodiment of ananalytical cell 114 is shown. The cell 114 has a block-like shape with asubstantially rectangular cross section having a length, l, measured inFIG. 2A along the z direction, which is substantially greater than itsdepth, d, measured along the x direction. The cell 114 includes threeconduits 118 having a substantially square cross sectional shape withequal height h and width w. The longitudinal axes of the conduits 118are substantially parallel to one another at a substantially equalpitch, p, and the conduits are arranged in a substantially coplanararray. Each conduit 118 is filled with a migration medium 120, which istypically a polymeric gel such as, for example, polyacrylamide.

In the embodiment shown in FIG. 2A the cell 114 includes a first wall122 with a first internal surface 124, as well as a substantiallyparallel and opposed second wall 126 with a second internal surface 128facing the first internal surface 124. The cell 114 further includes athird wall 130 that is generally normal to the planes of the first andsecond walls 122, 126. The third wall 130 has an internal surface 132.Any of the internal surfaces 124, 128 and 132 may be mirrored or atleast partially reflective to reflect light back into the cell 114.Preferably, at least part of the surface 132 is a mirror.

A light source 112, typically a laser, emits a light beam 113 having anoptical axis along the z direction and generally in the plane of theconduits 118. The light source 112 is a distance s_(z) from the cell114, and the light beam 113 enters the cell 114 at a fourth face 134 andtravels along the z direction a defined distance, referred to herein asthe atrium, a, until it reaches the first conduit in the array.

Light rays entering the cell 114 are internally reflected and remainconfined to the cell 114 to allow substantially uniform illumination ofall the conduits 118 in the array. Internal reflection in the cell 114is achieved by, for example, selection of materials with appropriaterefractive indices at the beam wavelength for the cell 114, themigration medium 120 and the surrounding medium 140 that is adjacent toat least one wall of the cell 114. Preferably, to achieve the mostuniform illumination of all the conduits in the array, the refractiveindices of the cell 114 and the migration medium 120 should match, or atleast be as similar as possible. This reduces the diffusive effect ofthe surfaces encountered by the incoming light rays. The cell 114 ispreferably made of a material that is transparent or translucent at thewavelength of the light emitted by the light source 112 and has lowbackground fluorescence at the wavelength(s) of the samplefluorophor(s). The cell 114 is typically a block of glass or plastic,although one skilled in the art could select a wide variety ofmaterials, depending on the wavelength emitted by the source 112, therefractive indices of the migration medium 120 and the surroundingmedium 140, and the fluorescence properties of the material. Suitablematerials for the cell 114 include, for example, fused silica glass,borosilicate glass, polycarbonate, polymethylmethacrylate,polymethylpentene, and cycloolefin copolymers.

The substantial internal reflection in the cell 114 is also achieved byselecting the shape and dimensions (length (l) and depth (d)) of thecell. The length and depth of the cell 114 illustrated in FIG. 2A areselected to provide a block-like shape, but many other shapes and lengthand/or depth variations may be used for the cell 114 depending on theintended application. For example, in a block like shape the overalllevel of illumination of the array typically decreases as the depth d ofthe cell increases. However, as the depth d decreases to approximatelythe dimension of the conduits, the illumination of the conduits nearestthe light source will be significantly greater than the illumination ofthe conduits farthest from the light source, i.e. the illuminationprofile of the array will be more non-uniform. For example, for roundcross section capillaries having an outside diameter of 120 μm spaced ata pitch of 240 μm in a cell of 200 μm depth, illumination varies about25% across a 104 capillary array. If the thickness of the cell isincreased to 300 μm, the variation in illumination is reduced to about6%, but at a loss of intensity of about 25%. Therefore, in addition tothe materials considerations discussed above, the overall dimensions ofthe cell may be selected to provide a predetermined illumination leveland illumination profile required for a particular assay or a particulardetector sensitivity level.

The overall shape of the cell 114 may also vary widely depending on thelevel of illumination and the illumination profile desired. For example,FIG. 2B shows a cell 114A with a generally trapezoidal cross sectionalshape. The cell 114A includes three conduits 118A having a substantiallysquare cross sectional shape with an equal height h and width w. Thelongitudinal axes of the conduits 118A are substantially parallel to oneanother at a substantially equal pitch, and the conduits are arranged ina substantially coplanar array. Each conduit 118A is filled with amigration medium 120A.

The cell 114A includes a first wall 122A with a first internal surface124A, as well as an opposed second wall 126A with a second internalsurface 128A facing the first internal surface 124A. The first wall 122Aand the second wall 126A gradually diverge at angles θ1 and θ2,respectively. The cell 114A further includes a third wall 130A thatpreferably has a reflective internal surface 132A. A light source 112Aemits a light beam 113A having an optical axis along the z direction andgenerally in the plane of the conduits 118A and substantially normal tothe longitudinal axes thereof. The light beam 113A enters the cell 114Aat a fourth face 134A. The face 134A has a depth d₁ that is less thanthe depth d₂ of the opposed face 130A. This trapezoidal cross-sectionalshape tends to recapture light that normally would be refracted out ofthe cell 114A, which tends to provide more uniform illumination of theconduits farthest from the light source 112A. The trapezoidal shapeprovides more options when, for example, the refractive index of thecell 114A or the refractive index of the surrounding medium are limitedto particular materials, or when there is a large refractive indexmismatch between the cell 114A and the migration medium 118A.

The refractive index difference at the interface between the cell andthe surrounding medium confines the light from the light source to thebody of the cell. The surrounding medium is preferably air. However, therefractive index of the surrounding medium may also be selected toprovide a particular level of illumination or illumination profile, andmay have an impact on the materials selected for the cell, as well asits dimensions. For example, the cell 114 may be placed in a liquid orsolid medium with a selected index of refraction, which may provide moreflexibility in the selection of materials for the cell and the migrationmedium for a particular assay application or to adapt to a particulardetector's dynamic range.

Referring to FIG. 3, representative light rays 113A and 113B are emittedby the decollimated source 112 and enter the cell 114 through the fourthface of the cell 134. For example, the ray 113B is initially reflectedat the first internal surface 128, illuminates the third conduit 118C inthe array, and is reflected back into the cell at the reflective thirdinternal surface 132. Following reflection at the third internal surface132, the ray 113B is again reflected at the second internal surface 124,illuminates the first conduit 118A in the array, and exits the cell 114at the fourth face 134. The internal reflection of the surrounding cell114 allows very efficient use of the light energy entering the cell tomore uniformly illuminate all conduits in the array.

Referring to FIG. 4, an alternate embodiment of the invention is shownwith a two-part fluorescence cell 150. The cell 150 includes amicrostructured substrate 152 and a substantially flat cover 154. Thecover 154 may be made of the same material as the substrate 152, or maybe made of a different material. The substrate 152 has machined orembossed therein an array of microgrooves 156. The longitudinal axes ofthe microgrooves 156 are substantially parallel, and the microgroovesare substantially uniform and coplanar in the array. The microgrooves156 are filled with a migration medium 158. When the cover is moved inthe direction of arrow A and placed on the substrate 152, the cell 150becomes a lightguide. Light 162 from a source 160 that enters thesubstrate 152 is internally reflected at the interior surfaces of thesubstrate 152 and the cover 154 to substantially uniformly illuminatethe microgrooves 156 in the array. In an alternate embodiment not shownin FIG. 4, both the substrate and the cover may be microstructured toform a wide variety of cross sectional shapes for the microgrooves 156.

As noted above, many current electrophoresis devices use capillaryarrays for high throughput analysis procedures. Referring to FIG. 5, anarray of capillaries may be inserted into a lightguide structure tocreate an analytical cell that substantially enhances the uniformity ofillumination of the individual capillaries in the array. In anelectrophoresis analysis system 210 shown in FIG. 5, a coating 211 isremoved from a series of capillaries 218 filled with a migration medium220. The stripped, bare ends of the capillaries 218 are inserted intoappropriately formed passages 215 in a block-like lightguide cell 214 toform a substantially coplanar array. The longitudinal axes of thecapillaries 218 are substantially parallel. A light beam 213 emittedfrom a source 212 enters the cell 214 to uniformly illuminate thecapillaries 218 and stimulating fluorescence from the fluorescentlylabeled analytes passing through the cell. This fluorescence is detectedby a detector (not shown) to obtain analytical data regarding theanalytes in the capillaries 218.

Referring to FIG. 6, a cross-sectional view of an embodiment of afluorescence cell 214 is shown. The cell 214 has a block-like shape witha substantially rectangular cross section having a length, l, measuredin FIG. 6 along the z direction, which is substantially greater than itsdepth, d, measured along the x direction. The cell 214 includes threecapillaries 218 having a substantially circular cross sectional shapewith a selected inside diameter (ID) and outside diameter (OD). Thelongitudinal axes of the capillaries 218 are substantially parallel toone another at a substantially equal pitch, p, and the capillaries arearranged in a substantially coplanar array. Each capillary 218 is filledwith a migration medium 220, which is typically a polymeric gel.

The cell 214 includes a first wall 222 with a first internal surface224, as well as a substantially parallel and opposed second wall 226with a second internal surface 228 facing the first internal surface224. The cell 214 further includes a third wall 230 that is generallynormal to the planes of the first and second walls 222, 226. The thirdwall 230 has an internal surface 232. Any of the internal surfaces 224,228 and 232 may be mirrored or at least partially reflective to reflectlight back into the cell 214. Preferably, at least part of the surface232 is a mirror (See also FIG. 5).

A light source 212, typically a laser, emits a light beam 213 having anoptical axis along the z direction and generally in the plane of thecapillaries 218. The light source 212 is a distance s_(z) from the cell214, and the light beam 213 enters the cell 214 at a fourth face 234 andtravels along the z direction a defined distance, the atrium, a, untilit reaches the first capillary in the array.

Light rays entering the cell 214 are internally reflected and remainconfined to the cell to allow substantially uniform illumination of allthe capillaries in the array. Substantial internal reflection in thecell 214 results from selection of materials with appropriate refractiveindices at the beam wavelength for the cell 214, the capillaries, themigration medium 220, and the surrounding medium 240. Preferably, toachieve the most uniform illumination of all the capillaries in thearray, the refractive indices of the cell 214, the capillaries 218, andthe migration medium 220 should match, or at least be as similar aspossible, to reduce the diffusive effect of the surfaces encountered bythe incoming light rays. The cell 214 is typically a block of glass orplastic, although one skilled in the art could select a wide variety ofmaterials, depending on the wavelength emitted by the source 212, therefractive indices of the capillaries 218, the migration medium 220, thesurrounding medium 240, and the fluorescence properties of the cellmaterial. Suitable materials include fused silica glass and borosilicateglass.

Referring to FIG. 7, a representative light rays 213A and 213B areemitted by the decollimated source 212 and enter the cell 214 throughthe fourth face of the cell 234. The ray 213A is initially reflected atthe first internal surface 224, illuminates the second capillary 218B inthe array, and is reflected back into the cell at the reflective secondinternal surface 228 and the reflective third internal surface 232.Following reflection at the third internal surface 232, the ray 213A isagain reflected at the first internal surface 124, illuminates thesecond capillary 218B in the array, is reflected at the second internalsurface 228, and exits the cell through the wall 234.

The internal reflection of the surrounding cell 214 allows veryefficient use of the light energy entering the cell to more uniformlyilluminate all capillaries in the array. In contrast to conventionaldevices, the flexibility provided by internal reflection also allows awide range of capillary inside and outside diameters. As a general rule,the design considerations discussed above with respect to cells withconduits also apply to cells using capillaries to retain the migrationmedium. However, the walls of the capillaries typically serve as anintegral part of the lightguiding portion of the cell, particularly iftheir refractive indices are well matched with the refractive indices ofthe cell and the migration medium.

Referring to FIG. 8, an alternate embodiment of the invention is shownwith a two-part analytical cell 250. The cell 250 includes amicrostructured substrate 252 and a corresponding microstructured cover254. The substrate 252 and the cover 254 have formed therein an array ofmicrogrooves 256. The longitudinal axes of the microgrooves 256 aresubstantially parallel, have arcuate cross sections, and aresubstantially uniform and coplanar in the array. In the microgrooves 256are placed capillaries 257, each filled with a migration medium 258.When the cover is moved in the direction of arrow A and placed on thesubstrate 252, the cell 250 becomes a lightguide. Light 262 from asource 260 that enters the substrate 252 is substantially internallyreflected at the interior surfaces of the substrate 252 and the cover254 to substantially uniformly illuminate the capillaries 257 in thearray.

The lightguiding properties of the cells described above allow forconsiderable variation in array design. The internal reflection of thecell provides sufficient illumination of the capillaries or microgrooves(also referred to generally herein as conduits) in the array, even ifindividual conduits are displaced by small amounts from their nominalpositions. The conduits need not be placed at an even pitch, even intheir nominal positions. The lightguiding properties of the cell makethe arrays of the invention robust against inaccuracies in conduitplacement during cell manufacture. However, referring to FIG. 9, a cell314 with a close packed coplanar arrangement of conduits 318, with allconduits touching each other in the plane of the array, appears toprovide the highest and most uniform illumination. In fact, thelightguiding properties of the cells described above provide uniformconduit illumination even for non-planar, close-packed arrangements. Forexample, the cell 414 illustrated in FIG. 10 includes capillaries 418 ina staggered, close-packed arrangement. This allows more conduits to beplaced into a given fixed field of view of a detector such as a CCDcamera, which maximizes the number of samples that can be analyzedsimultaneously with one instrument.

The lightguiding properties of the cells described above alsoaccommodate a wide variety of conduit cross sectional shapes. Manydifferent conduit cross sectional shapes are possible, such as circles,squares, rectangles, triangles, ellipses, and the like. However,conduits with square cross sections, including microgrooves andcapillaries, are preferred. The square cross sectional shape appears toprovide the most uniform illumination of the array, at least when theincoming light is directed in the plane of the array and normal to thelongitudinal axes of the conduits. While not wishing to be bound by anytheory, the square conduit is believed to present a flat face to theincoming light beam, which minimizes reflection and refraction out thecell. For example, referring to FIG. 11, a cell 514 is shown having anarray of capillaries 518 with square cross sectional shapes. To takeadvantage of this optimized conduit shape for commonly used capillarieswith a circular cross section, FIG. 12 shows a cell 614 constructed as amonolithic block with square internal microgrooves 618. The cell 614includes recesses 619 with a circular cross section and a matingshoulder 621 to allow secure attachment of capillaries 623 to the cell614. This design exploits the advantages of microgrooves arrays in thedetection region of the cell 614, which has fewer surfaces and a squarecross sectional shape to minimize refraction, but preserves the glasscapillary format for analytical separations.

To provide the most uniform illumination of the conduits in the cellarray, it is preferred that the light beam entering the array be shapedand decollimated. As shown in FIG. 13A, a source 712 emits a beam 713spread in the direction in the plane of the array and generally normalto the longitudinal axes of the conduits (See, for example, the x axisof FIGS. 2-3.), by an amount referred to herein as an angular valueα_(x). An optimal range of the value α_(x), defined as the standarddeviation of a Gaussian distribution of the launch angle, provides ahomogenized light front that propagates down the cell. If α_(x) is toosmall, refraction at the first conduit encountered by the beameffectively “shadows” a number of the adjacent conduits in the array,which significantly decreases the illumination of the “downstream”conduits. Above an optimal value of α_(x), refraction out of the cellbecomes dominant, and the overall intensity received by each conduitappears to decrease monotonically as α_(x) increases. For example, for acell in air with a depth of 200 μm and circular capillaries with adiameter of 120 μm and placed at a pitch of 240 μm, an optimal value ofα_(x) appears to be a divergence half angle of about 5° to about 50°,preferably about 10° to about 20°. The value of α_(x) may also beexpressed in terms of a numerical aperture (NA) according to theequation NA=n sin(α_(x)), where n is the refractive index of thesurrounding medium. The preferred range of NA for the cell with a depthof 200 μm and circular capillaries with a diameter of 120 μm and placedat a pitch of 240 μm is about 0.09 to about 0.77, preferably about 0.17to about 0.34.

In addition, referring to FIG. 13B, beam divergence in the y direction,in the plane of the array (See, for example, the y axis of FIGS. 2-3.),referred to herein as an angular value α_(y), is preferably made smallto minimize simultaneous excitation of multiple analytes, particularlyin the conduits farthest away from the source. An optimal value of α_(y)appears to be a divergence half angle of approximately 1° or less.

The decollimation of the beam may be accomplished in many differentways. For example, an optical train may be placed between the source andthe cell to provide the proper beam shape and divergence. In analternative shown in FIG. 14, the face 934 of the cell 914 may be shapedto be a plano-concave lens 935 with an appropriate radius of curvatureto provide proper beam divergence. In another alternative that would beexpected to be more tolerant of misalignment between the light sourceand the cell, a cell 1014 is shown in FIG. 15 that includes agrating-like face 1034 that diverges the light rays 1013 entering thecell. Or, in the alternative, a diffuser may be placed in the beam pathto generate divergence in the light rays entering the cell. Many otherdiverging cell face designs would be apparent to those of ordinary skillin the art.

In another embodiment shown in FIG. 16, the cell 1114 may be illuminatedwith a first light source 1112A and a second light source 1112B placedon the opposite side of the cell. The second light source 1112B emits alight beam 1113B that enters the cell 1114 through the third face 1130and has an optical axis that is substantially collinear with the opticalaxis of the beam 1113A. In this embodiment the interior surface 1132 ofthe third face 1130 is not reflective.

EXAMPLES Example 1

A cell was modeled with a microgrooves configuration similar to thatshown in FIGS. 2-4, using a polymer gel as the migration medium. Thecell had the dimensions and material properties shown in Table 1 below.

TABLE 1 Number of Microgrooves 104 Microgrooves Width w (μm) 50Microgrooves Height h (μm) 50 Pitch (μm) 240 Cell Depth (μm) 200 Atrium(mm) 2 Beam Radius (μm) 25 Beam Divergence α_(x) (deg, x 20 direction)Beam Divergence α_(y) (deg, y 1 direction) Source Distance (sz) 20 (μm)Index of Refraction of Cell 1.49 Index of Refraction of 1.41 MigrationMedium Index of Refraction of 1 Surrounding Medium Intrinsic Absorption0.004 Coefficient of Cell (1/mm) Intrinsic Absorption 0.004 Coefficientof Migration Medium (1/mm) Intrinsic Absorption 0 Coefficient ofSurrounding Medium (1/mm)

This cell design was optically modeled using ray tracing simulationswell known in the art. The results, which are shown in FIG. 17, areexpressed in units of relative illumination, defined as the fraction ofthe power each microgrooves would have absorbed had a 50 μm laser beamdirectly illuminated the microgrooves without reflection or refraction.The microgrooves were numbered sequentially from 1 to 104, withmicrogrooves 1 located nearest the light source. The results indicateextremely uniform illumination for all the microgrooves in the 104member array.

Example 2

A cell was modeled with capillaries in the general configuration shownin FIGS. 5-8, using a polymer gel as the migration medium. The cell hadthe dimensions and material properties shown in Table 2 below.

TABLE 2 Number of Capillaries 104 Capillaries ID (μm) 50 Capillaries OD(μm) 120 Pitch (μm) 240 Cell Depth (μm) 200 Atrium (mm) 2 Beam Radius(μm) 25 Beam Divergence α_(x) (deg, x 20 direction) Beam Divergenceα_(y) (deg, y 1 direction) Source Distance sz 20 (μm) Index ofRefraction of Cell 1.49 Index of Refraction of 1.41 Medium Index ofRefraction of 1.46 Capillaries Index of Refraction of 1 SurroundingMedium Intrinsic Absorption 0.004 Coefficient of Cell (1/mm) IntrinsicAbsorption 0.004 Coefficient of Migration Medium (1/mm) IntrinsicAbsorption 0.004 Coefficient of Capillaries (1/mm) Intrinsic Absorption0 Coefficient of Surrounding Medium (1/mm)

This cell design was optically modeled using well known ray tracesimulations and the criteria of Example 1. The results are shown in FIG.18. Very uniform illumination is achieved despite the plethora ofsurfaces in the system. Furthermore, this comes at a reasonable cost inintensity. Overall, the 104 capillaries in the array absorb only about0.34% of the total beam power.

Example 3

In this example the sensitivity of the cell performance was evaluatedwith regard to two common types of optical misalignment that may occurduring manufacture or operation of an analytical device. First, a cellsimilar to that of Example 2 was modeled. A baseline relativeillumination value was established using a laser light source that wasproperly aligned with the cell. Relative illumination was also computedfor a case where the light source was tilted about 20° away from theplane of the array. In addition, relative illumination was measured fora case where all 104 capillaries were randomly displaced from theirnominal locations by ±25 μm in either the x or z directions (See axes inFIG. 6). The results are shown in FIG. 19. Despite rather extremeexcursions from optimum optical alignment, neither intensity noruniformity appear to be significantly reduced.

Example 4

In this example the relative illumination intensity was evaluated withrespect to variations in the angular spread of the light beam in the xdirection (See axes in FIGS. 6 and 13), α_(x). Using the capillary arrayof Example 2, α_(x) was varied from 10° to 50°. The results are shown inFIG. 20. The results plotted in FIG. 20 indicate that if α_(x) is toosmall, refraction at the first conduit encountered by the beameffectively “shadows” a number of the adjacent conduits in the array,which significantly decreases illumination. Above an optimal value ofα_(x), the overall intensity received by each conduit appears todecrease monotonically as α_(x) increases.

Example 5

In this example the relative illumination values of capillaries withcircular cross sections are compared to those with square crosssections. First, the array of Example 2, which had 104 capillaries withcircular cross sections, was evaluated. Then a second cell was modeledwith 104 capillaries having square cross sections (See FIG. 11). Bothcells were evaluated using ray trace simulations well known in the art,and the results are shown in FIG. 21. As noted above, the flat faces ofthe square capillaries reduce out-of-plane refraction of incoming light,which enhances illumination.

Example 6

First, the relative illumination of the 104 capillary array of Example 2was evaluated using ray trace simulations well known in the art. Thisarray, as shown in FIGS. 5-8, included a reflective third interiorsurface 232. A second array identical to that of Example 2 except for anon-reflective third interior surface, was evaluated. A third array,similar to that shown FIG. 16, was modeled using two sources and anon-reflective interior surface 1132, and then evaluated using ray tracesimulations well known in the art. The results are shown in FIG. 22. Thesingle source device with a reflective third interior surface providedthe best levels of relative illumination, followed by the dual sourcedevice. The single source device without the reflective third interiorsurface provided relatively poor illumination to the downstreamcapillaries in the array.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An analytical device, comprising: a lightguide consisting of a solidblock of a transparent or a translucent material, wherein the lightguidecomprises an array of close-packed conduits extending therethrough suchthat all the conduits touch each other in a plane of the array, and amigration medium in the conduits; a first light source outside thelightguide, wherein the first light source emits a first light beam in afirst direction with an optical axis substantially coplanar with andnormal to the longitudinal axes of the conduits, a second light sourceoutside the lightguide, wherein the second light source emits a secondlight beam in a second direction substantially collinear with andopposite to the first direction; wherein the migration medium, thelightguide and a medium surrounding the lightguide have refractiveindices selected such that light emitted by the first and second lightsources is totally internally reflected within the lightguide toilluminate the migration medium in the conduits.
 2. The device of claim1, wherein the longitudinal axes of the conduits in the array aresubstantially parallel and coplanar.
 3. The device of claim 2, whereinthe longitudinal axes of the conduits in the array are at asubstantially equal pitch to one another.
 4. The device of claim 1,wherein the conduits have a substantially circular cross section.
 5. Thedevice of claim 1, wherein the conduits have a substantially squarecross section.
 6. The device of claim 1, wherein the conduits arecapillary tubes.
 7. The device of claim 1, further comprising a detectoroptically coupled with the lightguide.
 8. The device of claim 1, whereinat least one of the first and second light beams are decollimated. 9.The device of claim 1, wherein the beams from the first and second lightsources diverge in a direction normal to a plane containing theconduits.
 10. The device of claim 9, wherein at least one of the firstand second light beams has a divergence half angle of at least about 20°in a direction normal to a plane containing the conduits.
 11. The deviceof claim 9, wherein at least one of the first and second light beams hasa spread of no more than about 1° in a plane parallel to a planecontaining the conduits.
 12. The device of claim 1, wherein thelightguide is made of a glass selected from the group consisting offused silica and borosilicate.
 13. The device of claim 1, wherein thelightguide is a polymeric material selected from the group consisting ofpolycarbonate, polymethylmethacrylate, polymethylpentene, andcycloolefins.